On-line Solid-phase Extraction-liquid Chromatography-tandem Mass Research Articles

High-Throughput Analysis of Amphetamines in Blood and Urine with Online Solid-Phase Extraction-Liquid Chromatography—Tandem Mass Spectrometry

An automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS) method for the analysis of amphetamines in blood and urine was developed and validated. Chromatographic separation was achieved on a Nucleodur Sphinx RP column with an LC gradient (a mixture of 10 mM ammonium formate buffer and acetonitrile), ensuring the elution of amphetamine, methamphetamine, MDMA, MDA, MDEA, PMA, and ephedrine within 11 min. The method was fully validated, according to international guidelines, using only 100 and 50 microL of blood and urine, respectively. The method showed an excellent intra- and interassay precision (relative standard deviation < 11.2% and bias < 13%) for two external quality control samples (QC) for both matrices and three and two 'in house' QCs for blood and urine, respectively. Responses were linear over the investigated range (r(2) > 0.99, 2.5-400 microg/L for blood and 25-1000 microg/L for urine). Limits of quantification were determined to be 2.5 and 25 microg/L for blood and urine, respectively. Limits of detection ranged from 0.05 to 0.5 microg/L for blood and 0.25 to 2.5 microg/L for urine, depending on the compound. Furthermore, the analytes and the processed samples were demonstrated to be stable (in the autosampler for at least 72 h and after three freeze/thaw cycles), and no disturbing matrix effects were observed for all compounds. Moreover, no carryover was observed after the analysis of high concentration samples (15,000 microg/L). The method was subsequently applied to authentic blood and urine samples obtained from forensic cases, which covered a broad range of concentrations. The validation results and actual sample analyses demonstrated that this method is rugged, precise, accurate, and well-suited for routine analysis as more than 72 samples are analyzed non-stop in 24 h with minimum sample handling. The combination of the high-throughput online SPE and the well-known sensitivity and selectivity assured by MS-MS resulted in the elimination of the bottleneck associated with the sample preparation requirements and provided increased sensitivity, accuracy, and precision.

Simultaneous occurrence of nitrates and sulfonamide antibiotics in two ground water bodies of Catalonia (Spain)

Summary In the present work the occurrence of 19 selected sulfonamides, including one acetylated metabolite, was investigated in ground water samples taken from two ground water bodies in Catalonia (Plana de Vic and La Selva). Both include areas designated as nitrate vulnerable zones, according to Directive 91/676/EEC. A fully automated analytical methodology based on on-line solid-phase extraction–liquid chromatography-tandem mass spectrometry (on-line SPE–LC–MS/MS) was developed for this purpose. The high selectivity and sensitivity achieved (limits of detection between 0.005 and 0.8 ng/L) permitted to demonstrate the ubiquity of these antibiotics in both ground water bodies. Results showed a wide range of concentrations, from 0.01 ng/L up to 3460.57 ng/L. Since sulfonamides are related to livestock veterinary practices, they can be used as a specific indicator of manure contamination. However, the presence of sulfonamides appeared not to be directly related to the concentration of nitrates, as it is reflected on the low correlation coefficients found.

On-line coupling of solid-phase extraction to liquid chromatography–tandem mass spectrometry for the determination of macrolide antibiotics in environmental water

An automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) system was developed for the determination of macrolide antibiotics including erythromycin (ETM), roxithromycin (RTM), tylosin (TLS) and tilmicosin (TMC) in environmental water samples. A Capcell Pak MF Ph-1 column packed with restricted access material (RAM) was used as SPE column for the concentration of the analytes and clean-up of the sample. One milliliter water sample was injected into the conditioned SPE column and the matrix was washed out with 3 mL high purity water. By rotation of the switching valve, macrolides (MLs) were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase. The matrix effect was evaluated by the directly injection LC–MS and on-line SPE–LC–MS methods. The limits of detection (LODs) and limits of quantification (LOQs) obtained are in the range of 2–6 and 7–20 ng L −1, respectively, which means that the proposed method is suitable for trace analysis of MLs at low level concentration. The intra- and inter-day precisions are in the range of 2.9–7.2% and 3.3–8.9%, respectively. In the three fortified levels (20, 200 and 2000 ng L −1), recoveries of MLs ranging from 86.5% to 98.3% are obtained.

Analysis of 17 polar to semi-polar pesticides in the Ebro river delta during the main growing season of rice by automated on-line solid-phase extraction-liquid chromatography–tandem mass spectrometry

This work describes the optimization of a fully automated method based on on-line solid-phase extraction-liquid chromatography–electrospray–tandem mass spectrometry (SPE-LC–ESI-MS/MS) for the simultaneous determination of 17 medium to polar pesticides in water. The list of target analytes included organophosphates, triazines, phenylureas, anilides, chloroacetanilides, acidic herbicides and thiocarbamates. Detection limits achieved were below 5 ng/L for all compounds except metolachlor (12 ng/L), alachlor (17 ng/L), malathion (36 ng/L) and fenitrothion (50 ng/L). The method developed was used to investigate the occurrence of the target pesticides in a total of 52 water samples collected monthly (from May to August 2005) at 14 selected locations in the rice cultivation area of the Ebro river delta. The study showed high levels, in the μg/L range, of bentazone, MCPA, propanil, molinate and atrazine, in basically all the samples investigated. The remaining pesticides were present at lower levels (<0.1 μg/L) or only detected sporadically ( e.g. fenitrothion and malathion). The sampling campaign performed in July showed comparatively higher levels than the other three campaigns.

High-throughput on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry method for the simultaneous analysis of 14 antidepressants and their metabolites in plasma

A rapid, sensitive and fully automated on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma. Integration of the sample extraction and LC separation into a single system permitted direct injection of the plasma without prior sample pre-treatment. The applied gradient ensured the elution of all the examined drugs within 14 min and produced chromatographic peaks of acceptable symmetry. The total process time was 20 min and only 50 μL of plasma was required. Selectivity of the method was achieved by a combination of retention time and two precursor-product ion transitions for the non-deuterated compounds. The use of SPE was demonstrated to be highly effective and led to significant decreases in the interferences present in the matrix. Extraction was found to be both reproducible and efficient with recoveries >99% for all the analytes. The method showed excellent intra-assay and inter-assay precision (relative standard deviation (RSD) and bias <20%) for quality control (QC) samples spiked at a concentration of 40, 200 and 800 μg/L and the r 2 > 0.99 over the range investigated (10–1000 μg/L). Limits of quantification (LOQs) were estimated to be 10 μg/L. Furthermore, the processed samples were demonstrated to be stable for at least 48 h, except for clomipramine and norclomipramine, where a slight negative trend was observed, but did not compromise the quantification. The method was subsequently applied to authentic samples previously screened by a routine HPLC method with diode array detection (DAD).

Combined use of biomarkers and in situ bioassays in Daphnia magna to monitor environmental hazards of pesticides in the field

The aim of this investigation was to evaluate toxicity effects of pesticides in aquatic invertebrates using in situ bioassays with the local species, Daphnia magna. Investigations were carried out in the Delta del Ebro (northeast Spain) during the main growing season of rice (May-August). Measures of energy consumption (i.e., algal grazing) and of specific biochemical responses (biomarkers) were conducted in individuals transplanted at four stations, including a clean site upstream of the affected area and the three main channels that collect and drain the water from the rice fields into the sea. Seventeen pesticides were analyzed in water by on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry. The results obtained indicated high levels of pesticides in water, with peak values of 487 microg/L for bentazone, 8 microg/L for methyl-4-chlorophenoxyacetic acid, 5 microg/L for propanil, 0.8 microg/L for molinate, and 0.7 microg/L for fenitrothion. Measured biological responses denoted severe effects on grazing rates; a strong inhibition of cholinesterases and carboxylesterases, which are specific biomarkers of organophosphorous and carbamate pesticides; and altered patterns of the antioxidant enzyme catalase and the phase II metabolizing enzyme glutathione S-transferase. Correlation analysis with pesticide residue levels converted to toxic units relative to its acute 48-h median lethal concentration of D. magna indicated significant and negative coefficients between the dominant pesticide residues and the observed biological response, thus denoting a clear cause-and-effect relationship. The results emphasize the importance of considering specific (biomarkers) as well as more generalized and ecologically related (grazing) in situ responses to identify and evaluate biological effects of environmental contaminants in the field.

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma

In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-microL sample volume, which was only diluted with 200 microL of H2O (containing 500 ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6 min, subsequent analyses 3.3 min between injections) and has been validated in terms of the limit of detection (LOD, 2-70 ng/mL), lower limit of quantification (LLOQ, 78-156 ng/mL), linearity (R2, 0.9971-0.9989), linear concentration range (from LLOQ to 10,000 ng/mL), intra- and inter-day precision (< 13.5% at LLOQ, < 7.5% at high concentrations), proficiency testing accuracy (78-127%), laboratory internal accuracy (86-113%), recovery (60-110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.

Determination of three phthalate metabolites in human urine using on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry

An on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry (on-line SPE–HPLC–MS/MS) method was developed for the analysis of metabolites of three phthalate esters in human urine at the low nanogram per milliliter level. The recoveries were above 84.3% and relative standard deviations varied from 0.8 to 4.8%. The compounds along with their deuterated internal standards were detected in the negative ion mode by selective reaction monitoring and the accuracy of the method was improved by isotope dilution. Monobutyl phthalate was detected with median level of 22.5 ng/ml. The median levels for monobenzyl phthalate and monoethylhexyl phthalate were less than the limit of quantitation (LOQ). The on-line SPE–HPLC–MS/MS method allowed the possibility of determining these metabolites within a short time, with increased sensitivity and by using decreased amounts of sample and solvent.

Trace determination of priority pesticides in water by means of high-speed on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry using turbulent-flow chromatography columns for enrichment and a short monolithic column for fast liquid chromatographic separation

An integrated on-line SPE–HPLC–MS/MS system has been developed for the rapid analysis of various trace level priority pesticides in surface and drinking water. Eleven pesticides were included in this study, with various phenylureas, triazines and organophosphorous species among them. Use of turbulent-flow chromatography columns (TFC, 50×1 mm, 30–50 μm particle size) as extraction cartridges enables fast on-line SPE at high sampling flow-rate (5 ml/min). Polymeric and carbon based TFC columns (Oasis HLB, Cyclone, Hypercarb) allow complete extraction with good recoveries from water volumes up to 50 ml. On-line coupling to HPLC is performed with re-mixing of the organic TFC eluate with water in front of the analytical column to ensure efficient band focussing. For fast HPLC analysis, a short monolithic column is applied in combination with highly selective API–MS/MS detection. Matrix effects on the APCI–MS/MS signal were found to be reduced by the system to an acceptable minimum. Limits of detection, determined for 10-ml samples of river water were in the range between 0.4 and 13 ng/l typically, except trifluralin (approximately 280 ng/l), which is less susceptible to ionization under atmospheric pressure conditions. At an enriched water volume of 10 ml, the whole SPE–HPLC–MS/MS procedure requires less than 14 min. The method was successfully applied to the analysis of drinking and surface water samples taken from several sampling sites around the city of Leipzig, Germany. Concentrations measured (maximum: 16 ng/l simazine in river water) were far below the concentration limits scheduled by law.

Determination of quaternary ammonium pesticides by liquid chromatography–electrospray tandem mass spectrometry

A method for the direct determination of paraquat, diquat, chlormequat and difenzoquat in water samples, using an on-line solid-phase extraction–liquid chromatography–tandem mass spectrometry system was developed. No sample preparation was required and the detection limits were below the European Union maximum residue levels. The chromatographic separation was performed using an XTera MS C 8 column. The concentration of the ion pair reagent, the pH and the gradient elution were optimized to give high recoveries and good chromatographic resolution between quats. The detection was carried out using an ion trap as mass analyzer. Parameters such as the magnitude and duration of the resonant excitation voltage and the magnitude of the trapping RF voltage for full scan tandem mass spectrometry (MS–MS) experiments were studied to establish the optimal experimental conditions. Moreover, the accurate optimization of these parameters allowed MS–MS experiments of low mass ions, below m/z 200, providing unambiguous peak identification. Finally, the reproducibility of the proposed method was shown by good run-to-run and day-to-day precision values and its applicability to the determination of quats in drinking water was evaluated using spiked samples.